The detailed structural and energetic origins of sequence-specificity in the recognition and modification of DNA by proteins will be investigated using EcoRV endonuclease as the experimental system. A strategy encompassing directed modification of the enzyme and DNA, based on the high-resolution Xray cocrystal structures, will be used to test current hypotheses regarding the basis for discrimination. Specific questions to be addressed include the role of extensive enzyme and DNA conformational changes (induced fit), which are required to form the specific complex. The underlying basis for indirect readout of DNA sequence information will also be addressed. Rational strategies will be employed to expand the sequence specificity of the enzyme beyond the six base-pair cognate DNA site, and the database of known restriction endonuclease-DNA structures will be expanded by determination of several new enzymes. Creation of novel restriction endonucleases with expanded specificity will be extremely useful in aiding current efforts at large-scale physical mapping of the human genome. Since restriction endonucleases form an essential part of the recombinant DNA technology underlying all areas of biological research, the development of enzymes with new specificities will have a wide-ranging impact on the development of therapies for many human diseases.